Alcoholism is a progressive relapsing disorder, with relapse rates from 50-90% in spite of current treatments. The development of new treatment strategies requires an understanding of the neurobiological underpinnings of alcoholism. A primary source of relapse is the negative affect (i.e. anxiety and depression) associated with alcohol withdrawal. The bed nucleus of the stria terminalis, BNST, is a brain region implicated in this negative affect associated with alcohol withdrawal. Long-term adaptations in the BNST occur through the neurotransmitter glutamate and the NMDA receptor (NMDAR). In the BNST, my recent work illustrates that the acute and chronic actions of alcohol are caused by a particular NMDAR subunit (GluN2B). Further, chronic alcohol leads to an increase in transmission through these GluN2B-NMDARs at extrasynaptic locations. Signaling through extrasynaptic GluN2B-NMDARs has been connected to a number of pathological conditions; however, the mechanisms underlying the upregulation and relocation of GluN2B-NMDARs after chronic alcohol are unknown. In this proposal, I will be using a novel, highly specific discovery-based proteomic approach in conjunction with electrophysiological and biochemical techniques to identify alcohol-induced changes in GluN2B interacting proteins in synaptic and extrasynaptic locations. Preliminary work suggests that chronic alcohol may alter proteins that link NMDARs and signaling partners within the synapse (Homer, GKAPs, Shank, mGlur5, and PSD proteins). I propose that destabilization of these proteins by chronic alcohol allows for the relocalization o GluN2B-NMDARs (Aim 1). mGluR5 is another glutamate receptor that has been shown to modulate plasticity in the BNST. These receptors are also known mediators of metaplasticity (plasticity of NMDARs) and the developmental subunit shifts from GluN2A to GluN2B in other regions. Therefore, mGluR5 signaling is a strong candidate for the enhancement of GluN2B-NMDARs following chronic alcohol (Aim 2). While understanding these critical neuroadaptations in adults is key, clinical data demonstrates that the strongest predictor for alcohol dependence is adolescent alcohol use. Basic research finds many differential effects of alcohol between adolescents and adults; one of these being prolonged negative affect during withdrawal from chronic alcohol. The cortex and hippocampus (BNST inputs) undergo declines in NMDAR abundance and shifts in NMDAR subunit composition (GluN2B to 2A) during adolescence. In contrast, the BNST seems to retain high GluN2B into adulthood. This intersection of these differential developmental trajectories within the BNST is likely to culminate in unique regulation of excitatory transmission and plasticity during adolescence. Therefore, the independent (R00) phase of this proposal will investigate alcohol-driven dynamic regulation of NMDAR composition, localization, and signaling during adolescence.